Regulatory T cells (Treg cells) are essential for preventing autoimmunity and immune pathology, which are caused by effector T cells in the absence of Treg cells Immune interventions aimed at restraining effector T cells (to limit autoimmunity and immune pathology) or boosting immune responses (e.g. against tumours) require approaches that change the balance between Treg cells and effector T cells. Current approaches are slow, inefficient, rely on the use of bioactive peptides purified from biological sources, or on the expansion of pre-existing Treg cells.
Expression of the transcriptional regulator Foxp3, an intracellular protein and member of the forkhead/winged-helix family of transcriptional regulators, is characteristic of Treg cells.
Treatment of T cells with the bioactive peptide TGFbeta is known to induce de novo expression of Foxp3 (Chen et al., 2003 J. Exp. Med. vol 198, pp 1875-1886).
Certain in vivo immunisation protocols have been reported to induce Foxp3 expression in T cell receptor transgenic T cells (Kretschmer et al., 2005 Nature Immunol. vol 6 p 1219). This approach requires knowledge of both the specificity for antigen and the MHC restriction of the target T cells, so that the antigen used for immunisation can be selected accordingly. This condition is met by artificial experimental systems in which T cells carry transgenic T cell receptors. It is a problem with this approach that this condition is not met for naturally occurring autoimmune diseases.
The mTOR inhibitor rapamycin (sirolimus) has been used to manipulate the expansion of pre-existing Treg cells expressing Foxp3 (Zheng et al., 2003 Immunity vol 19 p 503; Battaglia et al., 2005 Blood vol 105 p 4743). However, such treatments have been shown not to induce the de novo expression of Foxp3, which is a problem. For example, Battaglia et al (ibid) states ‘The presence of CD4 CD25 Tr cells . . . in rapamycin-exposed T-cell cultures may be due to either a de novo induction of CD25 Tr cells from CD25 T cells or to a selective expansion of the naturally occurring CD4 CD25 FoxP3 Tr-cell subset already present in limited amounts at the beginning of the culture (ie, the 10% of CD4 CD25bright T cells usually found in a naive spleen). To address this question, CD4 T cells depleted of the CD25 Tr cells were cultured for 3 weeks in the presence or absence of rapamycin. In contrast to CD4 T cells (FIG. 3A), CD4 CD25 T cells activated in the presence of rapamycin gave rise to a population of T cells that failed to suppress cell proliferation in vitro (FIG. 4A). Accordingly, FoxP3 expression was enhanced in CD4 T cells exposed to rapamycin but not in CD4 CD25 rapamycin-treated T cells (FIG. 4B)’. Furthermore, the protocols employed rely on multiple rounds of in vitro stimulation over several weeks, which has the drawback of being very labour intensive. Negative effects of rapamycin on Treg cell numbers have also been reported in the art. Rapamycin is not a synthetic compound and can therefore suffer from problems of impurities and/or variation or formulation problems.
Thus, prior art techniques are typically operating via stimulation or activation of existing Tregs which can enhance Foxp3 expression in cells already expressing it, or are based on blocking non-Tregs, leading to expansion/selection or over-representation of Tregs in the population. These outcomes are the same as rapamycin treatment as noted above. Another example is the use of TGFbeta which typically enhances an already present level of Foxp3 expression. No such approaches lead to de novo Foxp3 expression/de novo Tregs.
Gene targeting of the PI3K isoenzyme p110delta results in increased Treg cell numbers in the thymus, but in decreased Treg cell numbers in peripheral lymphoid organs. This is accompanied by inflammatory bowel disease, which is often linked to Treg cell deficiencies. No clear conclusions about a relationship between PI3K signalling and Foxp3 expression can be drawn from these studies.
Inhibitors of the PI3K isoenzyme p110gamma are under evaluation for the treatment of autoimmune diseases based on mechanisms distinct from Treg cells.
WO2004/032867A3 discloses molecules preferentially associated with effector T cells or regulatory T cells and methods of their use. This document presents population level studies. PI3K inhibitors are mentioned on page 76 of this document and in FIG. 23A. The effects on Foxp3 expression (if any—high doses of inhibitor according to this document are probably cytotoxic and the low doses arguably show no significant effect) appear to be due to an expansion of Foxp3 expressing cells, or an enhancement in expression of Foxp3 in Foxp3 expressing cells. There is no evidence of de novo Foxp3 expression in the teachings of WO2004/032867A3. The underlying principle of this document is in attempting to tilt the balance of the immune system, for example to try to generally reduce autoimmunity or to try to generally enhance responses. There is no teaching of, and no suggestion towards, making new Tregs. Their method at best teaches that resting cells (PBL) are simultaneously activated (CD3/CD28) and inhibited (LY294002), i.e. activation and stimulation at the same time. In common with other prior art studies (e.g. Battaglia (ibid.)), and in common with the inventors' own studies, such treatments do not generate de novo Foxp3 expression/de novo Tregs.
US2004/0072766 discloses methods for modulating T cell responses by manipulating intracellular signal transduction. The methods disclosed in this document involve the addition of stimulators and inhibitors together i.e. at once or simultaneously. No de novo Foxp3 expression is generated in this approach.
Foey et al (Arthritis Res 2002 vol 4 pp 64-70) disclose that cytokine stimulated T cells induce macrophage IL-10 production dependent on phosphatidyl inositol 3-kinase and p70S6K and the implications for rheumatoid arthritis. This relates to the study of macrophages in the presence of T cells. The T cells were fixed to separate the effects before and after contact. Thus, the cells are fixed and no longer alive when the PI3K inhibitors are added in these methods. Thus no de novo Foxp3 expression is produced by these methods.
Breslin et al disclose that rapamycin and LY294002 co-operate to inhibit T cell proliferation. This example, in common with other studies, involves exposing T cells to inhibitors in order to study aspects of their biology such as proliferation. The inhibitors are added before stimulation, typically at least 30 minutes prior to stimulation. Cell numbers or other parameters are then examined. No de novo Foxp3 expression is generated by such techniques.
US2005/0261317 disclose inhibitors of human PI3K delta. This document merely examines certain neutrophils, B cells and certain exocytotic cells, and is not connected with T cells or Tregs. The data presented are merely aimed at validating that the PI3K inhibitors disclosed actually block the relevant functions.
US2004/0126781 discloses methods of preventing immune-mediated abortion by inhibiting a CD28-mediated costimulatory signal. These methods involve blocking of CD28 with soluble ligand. Blocking of CD28 with soluble ligand is clearly mutually exclusive with stimulation/activation of T cells.
The present invention seeks to overcome problem(s) associated with the prior art.